Summary: Evolutionary Developmental Biology

1. Evolution is caused by the inheritance of changes in development. Modifications of embryonic
or larval development can create new phenotypes that can then be selected.

2. Darwin's concept of "descent with modification" explained both homologies and adaptations.
The similarities of structure were due to common ancestry (homology), while the modifications
were due to natural selection (adaptation to the environmental circumstances).

3. The Urbilaterian ancestor can be extrapolated by looking at the developmental genes common
to invertebrates and vertebrates and which perform similar functions. These include the Hox
genes that specify body segments, the tinman gene that regulates heart development, the Pax6
gene that specifies those regions able to form eyes, and the genes that instruct head and tail
formation.

4. Changes in the targets of Hox genes can alter what the Hox genes specify. The Ubx protein, for instance, specifies halteres in flies and hindwings in butterflies.

5. Changes of Hox gene expression within a region can alter the structures formed by that region. For instance, changes in the expression of Ubx and abdA in insects regulate the production of prolegs in the abdominal segments of the larvae.

6. Changes in Hox gene expression between body regions can alter the structures formed by that
region. In crustaceans, different Hox expression patterns enable the body to have or to lack maxillipeds on its thoracic segments.

7. Changes in Hox gene expression are correlated with the limbless phenotypes in snakes.

8. Changes in Hox gene number may allow Hox genes to take on new functions. Large changes
the numbers of Hox genes correlate with major transitions in evolution.

9. Duplications of genes may also enable these genes to become expressed in new places. The
formation of new cell types may result from duplicated genes whose regulation has diverged.

10. In addition to structures being homologous, developmental pathways can be homologous.
Here, one has homologous proteins organized in homologous ways. These pathways can be used
for different developmental phenomena in different organisms and within the same organism.

11. Deep homology results when the homologous pathway is utilized for the same function in
greatly diverged organisms. The instructions for forming the central nervous system and for
forming limbs are possible examples of deep homology.

12. Modularity allows for parts of the embryo to change without affecting other parts.

13. The dissociation of one module from another is shown by heterochrony (changing in the
timing of the development of one region with respect to another) and by allometry (when
different parts of the organism grow at different rates).

14. Allometry can create new structures (such as the pocket gopher cheek pouch) by crossing a
threshold.

15. Duplication and divergence are important mechanisms of evolution. On the gene level, the
Hox genes, the Distal-less genes, the MyoD genes, and many other gene families started as single genes. The diverged members can assume different functions.

16. Co-option (recruitment) of existing genes and pathways for new functions is a fundamental
mechanism for creating new phenotypes. One such recruitment is the limb development pathway being used to form eyespots in butterfly wings.

17. Developmental modules can include several tissue types such that correlated progression
occurs. here, a change in one portion of the module causes changes in the other portions. When
skeletal bones change, the nerves and muscles serving them also change.

18. Tissue interactions have to be conserved, and if one component changes, the other must. If a
ligand changes, its receptor must change. Reproductive isolation may result from changes in
sperm or egg proteins.

19. Developmental constraints prevent certain phenotypes from occurring. Such restraints may be physical (no rotating limbs), morphogenetic (no middle finger smaller than its neighbors), or
phyletic (no neural tube without a notochord).

20. The Hsp90 protein enables cells to accumulate genes that would otherwise give abnormal

Why Clone Mammals?

Given that we already knew from amphibian studies in the 1960s that nuclei were pluripotent, why clone mammals? Many of the reasons are medical and commercial, and there are good reasons why these techniques were first developed by pharmaceutical companies rather than at universities. Cloning is of interest to some developmental biologists who study the relationships between the nucleus and cytoplasm during fertilization or who study aging (and the loss of totipotency that appears to accompany it), but cloned mammals are of special interest to those people concerned with protein pharmaceuticals. Protein drugs such as human insulin, protease inhibitor, and clotting factors are difficult to manufacture. Due to immunological rejection problems, the human proteins are usually much better tolerated by patients than proteins from other animals. So the problem becomes how to obtain large amounts of the human protein. One of the most efficient ways of producing these proteins is to insert the human genes encoding them into the oocyte DNA of sheep, goats, or cows. Animals containing a gene from another individual (often of a different species) a transgene are called transgenic animals. A transgenic female sheep or cow might not only contain the gene for the human protein, but might also be able to express the gene in her mammary tissue and thereby secrete the protein in her milk. Thus, shortly after the announcement of Dolly, the same laboratory announced the birth of Polly (Schnieke et al. 1997). Polly was cloned from transgenic fetal sheep fibroblasts that contained the gene for human clotting factor IX, a gene whose function is deficient in hereditary hemophilia.


Producing transgenic sheep, cows, or goats is not an efficient undertaking. Only 20% of the treated eggs survive the technique. Of these, only about 5% express the human gene. And of those transgenic animals expressing the human gene, only half are female, and only a small percentage of these actually secrete a high level of the protein into their milk. (And it often takes years for them to first produce milk). Moreover, after several years of milk production, they die, and their offspring are usually not as good at secreting the human protein as the originals. Cloning would enable pharmaceutical companies to make numerous copies of such an "elite transgenic animal," all of which should produce high yields of the human protein in their milk. The medical importance of such a technology would be great, since such proteins could become much cheaper for the patients who need them for survival. The economic incentives for cloning are therefore enormous (Meade 1997).

Cloning mammals

In 1997, Ian Wilmut announced that a sheep had been cloned from a somatic cell nucleus from an adult female sheep. This was the first time that an adult vertebrate had been successfully cloned from another adult. To do this, Wilmut and his colleagues 1997 took cells from the mammary gland of an adult (6-year-old) pregnant ewe and put them into culture.

The culture medium was formulated to keep the nuclei in these cells at the resting stage of the cell cycle (G0). They then obtained oocytes (the maturing egg cell) from a different strain of sheep and removed their nuclei. The fusion of the donor cell and the enucleated oocyte was accomplished by bringing the two cells together and sending electrical pulses through them. The electric pulses destabilized the cell membranes, allowing the cells to fuse together. Moreover, the same pulses that fused the cells activated the egg to begin development. The resulting embryos were eventually transferred into the uteri of pregnant sheep. Of the 434 sheep oocytes originally used in this experiment, only one survived: Dolly

DNA analysis confirmed that the nuclei of Dolly's cells were derived from the strain of sheep from which the donor nucleus was taken (Ashworth et al. 1998; Signer et al. 1998). Thus, it appears that the nuclei of adult somatic cells can be totipotent. No genes necessary for development have been lost or mutated in a way that would make them nonfunctional. This result has been confirmed in cows (Kato et al. 1998) and mice (Wakayama et al. 1998). In mice, somatic cell nuclei from the cumulus cells of the ovary were injected directly into enucleated oocytes. These renucleated oocytes were able to develop into mice at a frequency of 2.5% . Interestingly, nuclei from other somatic cells (such as neurons or Sertoli cells) that are similarly blocked at the G0 stage did not generate any live mice. Cumulus cell nuclei from cows have also directed the complete development of oocytes into mature cows

The split between embryology and genetics

Morgan's evidence provided a material basis for the concept of the gene. Originally, this type of genetics was seen as being part of embryology, but by the 1930s, genetics became its own discipline, developing its own vocabulary, journals, societies, favored research organisms, professorships, and rules of evidence. Hostility between embryology and genetics also emerged. Geneticists believed that the embryologists were old-fashioned and that development would be completely explained as the result of gene expression. Conversely, the embryologists regarded the geneticists as uninformed about how organisms actually developed and felt that genetics was irrelevant to embryological questions. Embryologists, such as Frank Lillie, Ross Granville Harrison (1937), Hans Spemann (1938), and Ernest E. Just (1939), claimed that there could be no genetic theory of development until at least three major challenges had been met by the geneticists:

1. Geneticists had to explain how chromosomes which were thought to be identical in every cell of the organism produce different and changing types of cell cytoplasms.

2. Geneticists had to provide evidence that genes control the early stages of embryogenesis. Almost all the genes known at the time affected the final modeling steps in development (eye color, bristle shape, wing venation in Drosophila). As Just said (quoted in Harrison 1937), embryologists were interested in how a fly forms its back, not in the number of bristles on its back.

3. Geneticists had to explain phenomena such as sex determination in certain invertebrates (and vertebrates such as reptiles), in which the environment determines sexual phenotype.

Now that the necessity of relating the data of genetics to embryology is generally recognized and the Wanderlust of geneticists is beginning to urge them in our direction, it may not be inappropriate to point out a danger of this threatened invasion. The prestige of success enjoyed by the gene theory might easily become a hindrance to the understanding of development by directing our attention solely to the genom, whereas cell movements, differentiation, and in fact all of developmental processes are actually effected by cytoplasm. Already we have theories that refer the processes of development to gene action and regard the whole performance as no more than the realization of the potencies of genes. Such theories are altogether too one-sided.

Until geneticists could demonstrate the existence of inherited variants during early development, and until geneticists had a well-documented theory for how the same chromosomes could produce different cell types, embryologists generally felt no need to ground their science in gene action.

Nucleus or cytoplasm: Which controls heredity?

It is in Mendel's term, however, that we see how closely intertwined were the concepts of inheritance and development in the nineteenth century. Mendel's observations, however, did not indicate where these hereditary elements existed in the cell, or how they came to be expressed. The gene theory that was to become the cornerstone of modern genetics originated from a controversy within the field of physiological embryology. In the late 1800s, a group of scientists began to study the mechanisms by which fertilized eggs give rise to adult organisms. Two young American embryologists, Edmund Beecher Wilson and Thomas Hunt Morgan, became part of this group of "physiological embryologists," and each became a partisan in the controversy over which of the two compartments of the fertilized egg the nucleus or the cytoplasm controls inheritance. Morgan allied himself with those embryologists who thought the control of development lay within the cytoplasm, while Wilson allied himself with Theodor Boveri, one of the biologists who felt that the nucleus contained the instructions for development. In fact, Wilson 1896 declared that the processes of meiosis, mitosis, fertilization, and unicellular regeneration (only from the fragment containing the nucleus) "converge to the conclusion that the chromatin is the most essential element in development."* He did not shrink from the consequences of this belief. Years before the rediscovery of Mendel or the gene theory, Wilson 1895 noted, "Now, chromatin is known to be closely similar, if not identical with, a substance known as nuclein . which analysis shows to be a tolerably definite chemical composed of a nucleic acid (a complex organic acid rich in phosphorus) and albumin. And thus we reach the remarkable conclusion that inheritance may, perhaps, be effected by the physical transmission of a particular chemical compound from parent to offspring."

Some of the major support for the chromosomal hypothesis of inheritance was coming from the embryological studies of Theodor Boveri, a researcher at the Naples Zoological Station. Boveri fertilized sea urchin eggs with large concentrations of their sperm and obtained eggs that had been fertilized by two sperm. At first cleavage, these eggs formed four mitotic poles and divided into four cells instead of two. Boveri then separated the blastomeres and demonstrated that each cell developed abnormally, and in a different way, as a result of each of the cells having different types of chromosomes. Thus, Boveri claimed that each chromosome had an individual nature and controlled different vital processes.

Adding to Boveri's evidence, E. B. Wilson and Nettie Stevens demonstrated a critical correlation between nuclear chromosomes and organismal development: XO or XY embryos became male; XX embryos became female. Here was a nuclear property that correlated with development. Eventually, Morgan began to obtain mutations that correlated with sex and with the X chromosome, and he began to view the genes as being physically linked to one another on the chromosomes. The embryologist Morgan had shown that nuclear chromosomes are responsible for the development of inherited characters.

Life cycles and the evolution of developmental patterns

Traditional ways of classifying catalog animals according to their adult structure. But, as J. T. Bonner (1965) pointed out, this is a very artificial method, because what we consider an individual is usually just a brief slice of its life cycle. When we consider a dog, for instance, we usually picture an adult. But the dog is a "dog" from the moment of fertilization of a dog egg by a dog sperm. It remains a dog even as a senescent dying hound. Therefore, the dog is actually the entire life cycle of the animal, from fertilization through death.

The life cycle has to be adapted to its environment, which is composed of nonliving objects as well as other life cycles. Take, for example, the life cycle of Clunio marinus, a small fly that inhabits tidal waters along the coast of western Europe. Females of this species live only 2 3 hours as adults, and they must mate and lay their eggs within this short time. To make matters even more precarious, egg laying is confined to red algae mats that are exposed onlyduring the lowest ebbing of the spring tide. Such low tides occur on four successive days shortly after the new and full moons (i.e., at about 15-day intervals). Therefore, the life cycle of these insects must be coordinated with the tidal rhythms as well as the daily rhythms such that the insects emerge from their pupal cases during the few days of the spring tide and at the correct hour for its ebb.

One of the major triumphs of descriptive embryology was the idea of a generalizable life cycle. Each animal, whether an earthworm, an eagle, or a beagle, passes through similar stages of development. The major stages of animal development are illustrated in Figure. The life of anew individual is initiated by the fusion of genetic material from the two gametes the sperm and the egg. This fusion, called fertilization, stimulates the egg to begin development. The stages of development between fertilization and hatching are collectively called embryo genesis.

Throughout the animal kingdom, an incredible variety of embryonic types exist, but most patterns of embryo genesis are variations on five themes:

1. Immediately following fertilization, cleavage occurs. Cleavage is a series of extremely rapid mitotic divisions wherein the enormous volume of zygote cytoplasm is divided into numerous smaller cells. These cells are called blastomeres, and by the end of cleavage, they generally form a sphere known as a blastula.

2. After the rate of mitotic division has slowed down, the blastomeres undergo dramatic movements wherein they change their positions relative to one another. This series of extensive cell rearrangements is called gastrulation, and the embryo is said to be in the gastrula stage. As a result of gastrulation, the embryo contains three germ layers: the ectoderm, the endoderm, and the mesoderm.

3. Once the three germ layers are established, the cells interact with one another and rearrange themselves to produce tissues and organs. This process is called organogenesis. Many organs contain cells from more than one germ layer, and it is not unusual for the outside of an organ to be derived from one layer and the inside from another. For example, the outer layer of skin comes from the ectoderm, while the inner layer (the dermis) comes from the mesoderm. Also during organogenesis, certain cells undergo long migrations from their place of origin to their final location. These migrating cells include the precursors of blood cells, lymph cells, pigment cells, and gametes. Most of the bones of our face are derived from cells that have migrated ventrally from the dorsal region of the head.

4. As seen in Figure 2.1, in many species a specialized portion of egg cytoplasm gives rise to cells that are the precursors of the gametes (the sperm and egg). The gametes and their precursor cells are collectively called germ cells, and they are set aside for reproductive function. All the other cells of the body are called somatic cells. This separation of somatic cells (which give rise to the individual body) and germ cells (which contribute to the formation of a new generation) is often one of the first differentiations to occur during animal development. The germ cells eventually

DNA is the genetic material

The idea that genetic material is nucleic acid had its roots in the discovery of transformation in 1928. The bacterium Pneumococcus kills mice by causing pneumonia. The virulence of the bacterium is determined by its capsular polysaccharide. This is a component of the surface that allows the bacterium to escape destruction by the host. Several types (I, II, III) of Pneumococcus have different capsular polysaccharides. They have a smooth (S) appearance.

Each of the smooth Pneumococcal types can give rise to variants that fail to produce the capsular polysaccharide. These bacteria have a rough (R) surface (consisting of the material that was beneath the capsular polysaccharide). They are avirulent. They do not kill the mice, because the absence of the polysaccharide allows the animal to destroy the bacteria.

When smooth bacteria are killed by heat treatment, they lose their ability to harm the animal. But inactive heat-killed S bacteria and the ineffectual variant R bacteria together have a quite different effect from either bacterium by itself. Figure 1 shows that when they are injected together into an animal, the mouse dies as the result of a Pneumococcal infection. Virulent S bacteria can be recovered from the mouse postmortem.

In this experiment, the dead S bacteria were of type III. The live R bacteria had been derived from type II. The virulent bacteria recovered from the mixed infection had the smooth coat of type III. So some property of the dead type III S bacteria can transform the live R bacteria so that they make the type III capsular polysaccharide, and as a result become virulent (Griffith, 1928).


The component of the dead bacteria responsible for transformation was called the transforming principle. It was purified by developing a cell-free system, in which extracts of the dead S bacteria could be added to the live R bacteria before injection into the animal. Purification of the transforming principle in 1944 showed that it is deoxyribonucleic acid (DNA) (Avery et al., 1944).

The next step was to demonstrate that DNA provides the genetic material in a quite different system. Phage T2 is a virus that infects the bacterium E. coli. When phage particles are added to bacteria, they adsorb to the outside surface, some material enters the bacterium, and then ~20 minutes later each bacterium bursts open (lyses) to release a large number of progeny phage.

Figure2 illustrates the results of an experiment in 1952 in which bacteria were infected with T2 phages that had been radioactively labeled either in their DNA component (with 32P) or in their protein component (with 35S). The infected bacteria were agitated in a blender, and two fractions were separated by centrifugation. One contained the empty phage coats that were released from the surface of the bacteria; these consist of protein and therefore carried the 35S radioactive label. The other fraction consisted of the infected bacteria themselves.

Most of the 32P label was present in the infected bacteria. The progeny phage particles produced by the infection contained ~30% of the original 32P label. The progeny received very littleless than 1%of the protein contained in the original phage population. This experiment therefore showed directly that the DNA of parent phages enters the bacteria and then becomes part of the progeny phages, exactly the pattern of inheritance expected of genetic material (Hershey and Chase, 1952).

A phage (virus) reproduces by commandeering the machinery of an infected host cell to manufacture more copies of itself. The phage possesses genetic material whose behavior is analogous to that of cellular genomes: its traits are faithfully reproduced, and they are subject to the same rules that govern inheritance. The case of T2 reinforces the general conclusion that the genetic material is DNA, whether part of the genome of a cell or virus.

When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid enters the cells, and in some of them results in the production of new proteins. When a purified DNA is used, its incorporation leads to the production of a particular protein. Figure3 depicts one of the standard systems.

Although for historical reasons these experiments are described as transfection when performed with eukaryotic cells, they are a direct counterpart to bacterial transformation. The DNA that is introduced into the recipient cell becomes part of its genetic material, inherited in the same way as any other part. Its expression confers a new trait upon the cells (synthesis of thymidine kinase in the example of the figure). At first, these experiments were successful only with individual cells adapted to grow in a culture medium. Since then, however, DNA has been introduced into mouse eggs by microinjection; and it may become a stable part of the genetic material of the mouse (see 17 Rearrangement of DNA).

Such experiments show directly not only that DNA is the genetic material in eukaryotes, but also that it can be transferred between different species and yet remain functional.

The genetic material of all known organisms and many viruses is DNA. However, some viruses use an alternative nucleic acid, ribonucleic acid (RNA), as the genetic material. Although its chemical formula is slightly different from that of DNA, in these circumstances RNA exercises the same role. The general principle of the nature of the genetic material, then, is that it is always nucleic acid; in fact, it is DNA except in the RNA viruses.

Research
Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 98, 451-460.
Griffith, F. (1928). The significance of pneuomococcal types. J. Hyg. 27, 113-159.
Hershey, A. D. and Chase, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56.

Genes are DNA

The basic nature of the gene was defined by Mendel more than a century ago. Summarized in his two laws, the gene was recognized as a "particulate factor" that passes unchanged from parent to progeny. A gene may exist in alternative forms (alleles).

In diploid organisms, which have two sets of chromosomes, one copy of each chromosome is inherited from each parent. This is the same behavior that is displayed by genes. The equivalence led to the discovery that chromosomes in fact carry the genes.

The next step was the demonstration that each chromosome consists of a linear array of genes. Mendel’s laws predict that genes carried on different chromosomes will segregate independently (for additional description see supplement on Mendel's laws and dominance). However, genes that are on the same chromosome show linked inheritance. The basic observation is that genes on different chromosomes recombine at random from one generation to the next, whereas genes that are linked show a reduction in recombination, that is, they tend to stay together.

Genetic analysis allows the construction of a linkage map that connects all the genes carried by one chromosome (for additional description see supplement on Linkage and mapping). The genetic map of a linkage group corresponds to the physical existence of the chromosome.

On the genetic maps of higher organisms established during the first half of this century, the genes are arranged like beads on a string. They occur in a fixed order, and genetic recombination involves transfer of corresponding portions of the string between homologous chromosomes. The gene is to all intents and purposes a mysterious object (the bead), whose relationship to its surroundings (the string) is unclear.

The resolution of the recombination map of a higher eukaryote is restricted by the small number of progeny that can be obtained from each mating. Recombination occurs so infrequently between nearby points that it is rarely observed between different mutations in the same gene. By moving to a microbial system in which a very large number of progeny can be obtained from each genetic cross, it became possible to demonstrate that recombination occurs within genes. It follows the same rules that were previously deduced for recombination between genes.

Mutations within a gene can be arranged into a linear order, showing that the gene itself has the same linear construction as the array of genes on a chromosome. So the genetic map is linear within as well as between loci: it consists of an unbroken sequence within which the genes reside. This conclusion segues naturally into the modern view that the genetic material of a chromosome consists of an uninterrupted length of DNA that represents many genes.

A genome consists of the entire set of chromosomes for any particular organism, and therefore comprises a series of DNA molecules, each of which contains many genes. The ultimate definition of a genome is to determine the sequence of the DNA of each chromosome.